CN107417774A - A kind of Bt Pesticidal toxins of ivermectin coupling and its application - Google Patents
A kind of Bt Pesticidal toxins of ivermectin coupling and its application Download PDFInfo
- Publication number
- CN107417774A CN107417774A CN201710859935.9A CN201710859935A CN107417774A CN 107417774 A CN107417774 A CN 107417774A CN 201710859935 A CN201710859935 A CN 201710859935A CN 107417774 A CN107417774 A CN 107417774A
- Authority
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- China
- Prior art keywords
- ivermectin
- cry2ab
- insecticidal proteins
- succinoyl
- ivermectins
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
- 108700012359 toxins Proteins 0.000 title claims abstract description 36
- 239000003053 toxin Substances 0.000 title claims abstract description 34
- 231100000765 toxin Toxicity 0.000 title claims abstract description 34
- AZSNMRSAGSSBNP-UHFFFAOYSA-N 22,23-dihydroavermectin B1a Natural products C1CC(C)C(C(C)CC)OC21OC(CC=C(C)C(OC1OC(C)C(OC3OC(C)C(O)C(OC)C3)C(OC)C1)C(C)C=CC=C1C3(C(C(=O)O4)C=C(C)C(O)C3OC1)O)CC4C2 AZSNMRSAGSSBNP-UHFFFAOYSA-N 0.000 title claims abstract description 33
- 229960002418 ivermectin Drugs 0.000 title claims abstract description 33
- SPBDXSGPUHCETR-JFUDTMANSA-N 8883yp2r6d Chemical compound O1[C@@H](C)[C@H](O)[C@@H](OC)C[C@@H]1O[C@@H]1[C@@H](OC)C[C@H](O[C@@H]2C(=C/C[C@@H]3C[C@@H](C[C@@]4(O[C@@H]([C@@H](C)CC4)C(C)C)O3)OC(=O)[C@@H]3C=C(C)[C@@H](O)[C@H]4OC\C([C@@]34O)=C/C=C/[C@@H]2C)/C)O[C@H]1C.C1C[C@H](C)[C@@H]([C@@H](C)CC)O[C@@]21O[C@H](C\C=C(C)\[C@@H](O[C@@H]1O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C1)[C@@H](C)\C=C\C=C/1[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\1)O)C[C@H]4C2 SPBDXSGPUHCETR-JFUDTMANSA-N 0.000 title claims abstract description 32
- 230000000361 pesticidal effect Effects 0.000 title claims abstract description 28
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 14
- 230000008878 coupling Effects 0.000 title claims abstract description 11
- 238000010168 coupling process Methods 0.000 title claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 35
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 35
- 230000000749 insecticidal effect Effects 0.000 claims abstract description 34
- 239000007853 buffer solution Substances 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 230000004913 activation Effects 0.000 claims abstract description 6
- 239000000417 fungicide Substances 0.000 claims abstract description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 5
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims abstract description 5
- 230000003213 activating effect Effects 0.000 claims abstract description 3
- 239000002904 solvent Substances 0.000 claims abstract description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 8
- 101100275683 Bacillus thuringiensis subsp. kurstaki cry2Ab gene Proteins 0.000 claims description 7
- 150000002460 imidazoles Chemical class 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 229940014800 succinic anhydride Drugs 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 5
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 5
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 5
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 claims description 5
- 229940086542 triethylamine Drugs 0.000 claims description 5
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 230000015556 catabolic process Effects 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 3
- 238000005336 cracking Methods 0.000 claims description 3
- 238000006731 degradation reaction Methods 0.000 claims description 3
- 238000010612 desalination reaction Methods 0.000 claims description 3
- 238000011033 desalting Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000006166 lysate Substances 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 231100000614 poison Toxicity 0.000 claims description 3
- 239000002574 poison Substances 0.000 claims description 3
- 230000035939 shock Effects 0.000 claims description 3
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 3
- 230000009466 transformation Effects 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 2
- 150000003851 azoles Chemical class 0.000 claims description 2
- 239000000284 extract Substances 0.000 claims description 2
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 claims 1
- 229940092714 benzenesulfonic acid Drugs 0.000 claims 1
- 230000003139 buffering effect Effects 0.000 claims 1
- 238000000855 fermentation Methods 0.000 claims 1
- 230000004151 fermentation Effects 0.000 claims 1
- 210000002429 large intestine Anatomy 0.000 claims 1
- 241000500437 Plutella xylostella Species 0.000 abstract description 11
- 241000607479 Yersinia pestis Species 0.000 abstract description 5
- 230000001018 virulence Effects 0.000 abstract description 3
- -1 succinoyl Chemical group 0.000 abstract 3
- 241000238631 Hexapoda Species 0.000 description 9
- 239000003905 agrochemical Substances 0.000 description 6
- 235000013601 eggs Nutrition 0.000 description 6
- 239000000575 pesticide Substances 0.000 description 4
- 241000500441 Plutellidae Species 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 241000305071 Enterobacterales Species 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- WCXDHFDTOYPNIE-RIYZIHGNSA-N (E)-acetamiprid Chemical compound N#C/N=C(\C)N(C)CC1=CC=C(Cl)N=C1 WCXDHFDTOYPNIE-RIYZIHGNSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 239000005875 Acetamiprid Substances 0.000 description 1
- 241000724266 Broad bean mottle virus Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241000254173 Coleoptera Species 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 1
- 241000257303 Hymenoptera Species 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical class ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000021110 pickles Nutrition 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal protein (delta-endotoxin)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
Abstract
The present invention provides a kind of Bt Pesticidal toxins of ivermectin coupling, and its preparation method is as follows：4 " O succinoyl ivermectins, EDC and NHS are weighed, and is dissolved in DMSO solvents, 4 " O succinoyl ivermectins after must activating are stand-by；Take the Na of the Bt Cry2Ab insecticidal proteins configuration insecticidal proteins of BtCry2Ab containing 5mmol/L2CO3/NaHCO3Buffer solution, i.e. Bt Cry2Ab insecticidal proteins solution, it is stand-by；4 " O succinoyl ivermectins after the activation prepared are measured respectively and carry out coupling reaction with Bt Cry2Ab insecticidal proteins solution, obtain Bt Pesticidal toxins.Bt Pesticidal toxins of the present invention have stronger virulence, and it has more preferable insecticidal effect to diamondback moth, can be used as agricultural fungicides, i.e., have more preferable application prospect to preventing and treating agricultural pests.
Description
【Technical field】
The invention belongs to Agricultural pest control field, and in particular to the Bt Pesticidal toxins and its answer that a kind of ivermectin is coupled
With.
【Background technology】
In recent years, the fast development of agricultural sciences, pesticide control play a certain degree of facilitation.Agricultural chemicals can be effective
The harmful organisms such as agricultural disease, worm, grass, mouse are prevented and treated, ensure that this important step of agriculture increase harvest.But the production of disease drug resistance
It is raw, it is the problem that chemical pesticide or biological pesticide are not avoided that；Insect is exactly to overcome agricultural chemicals position to the agricultural chemicals resistance to the action of a drug
Point effect, is allowed to fail, is passivated.In actual applications, agricultural chemicals rotation administration, mixed pesticide are applied, increase agricultural chemicals to insect
The diversity of contact area, the sensitive strain for cultivating insect, increase ecological diversity to increase insect etc., substantially makes agriculture
Medicine action site keeps enough sensitiveness, seeks to overcome the means of resistance.Bioconjugation is to utilize Bioconjugation technology by two
Kind is toxin conjugated to form new toxin, and sensitiveness to keeping agricultural chemicals site etc. has great importance.
Dipel (Bacilus thuringiensis, abbreviation Bt) is the current maximum of output in the world, using most
For extensive a kind of microbial insecticide, its crystalline protein is to more than 570 Species of Lepidopterous Insect Pests and Diptera, Hymenoptera and coleoptera
Etc. tens of kinds insects have insecticidal toxicity.It is generally believed that insect is after feeding crystalline protein, by the midgut proteinase of itself by its
Digest and combine, go forward side by side with the acceptor in midgut epithelial cell brush edge film (BBMVs) again for the toxin protein of activity, toxin protein
In one step insertion film, hole or ion channel are formed, causes Ion leakage, Gut wall epithelial cells is destroyed, oozes enteron aisle Dissolve things inside
Enter haemocoele, cause septicemia, cause insect death；But the resistance to the action of a drug of insect causes the preventive effect of existing Bt toxin drastically to decline,
Therefore, Bt toxin is transformed, there is provided a kind of to have the Bt toxin compared with strong virus force be that practitioner institute is highly desirable.
【The content of the invention】
The technical problems to be solved by the invention are Bt Pesticidal toxins and its application for providing a kind of ivermectin coupling.
The present invention is that solve above-mentioned technical problem by the following technical programs：A kind of Bt desinsections poison of ivermectin coupling
Element, the concrete operation step of its preparation method are as follows：By 1:1.5:1.5 mol ratio weighs 4 "-O-succinoyl Yi Wei respectively
Rhzomorph, EDC and NHS, and be dissolved in DMSO solvents, activated with the carboxyl to 4 "-O-succinoyl ivermectins, obtain work
4 "-O-succinoyl ivermectins after change, it is stand-by；Take Bt Cry2Ab insecticidal proteins configuration Bt containing 5mmol/L Cry2Ab
The Na of insecticidal proteins2CO3/NaHCO3Buffer solution, i.e. Bt Cry2Ab insecticidal proteins solution, it is stand-by；The work prepared is measured respectively
4 "-O-succinoyl ivermectins and the Bt Cry2Ab insecticidal proteins solution after change, stirring carry out being coupled instead for 1 hour
Should, obtain Bt Pesticidal toxins.
Further, the preparation process of the Bt Cry2Ab insecticidal proteins is as follows：
(1) cry2Ab genes are converted to colibacillus engineering to obtain containing the big of cry2Ab genes by thermal shock method
Enterobacteria engineering bacteria, it is seeded to afterwards on LB solid mediums, LB solid mediums is placed in incubated 24h at 30 DEG C
Carry out actication of culture；
(2) the colibacillus engineering strain after activation is inoculated in LB fluid nutrient mediums, is placed in shaking flask 37 DEG C
Fermented and cultured, speed setting 180r/min, treat bacterium solution OD600After reaching 0.5,25 DEG C are adjusted to, 180r/min continues
Cultivate 24h；Then zymotic fluid obtained by fermented and cultured is placed in 4 DEG C, centrifuges 10min under the conditions of 10000r/min, after centrifugation terminates
Precipitation is taken to be resuspended in Na2CO3Lysate, the ultrasonic degradation 30min at 4 DEG C, 4 DEG C of solution, 10000r/min centrifugations after cracking
30min, take supernatant；
(3) supernatant obtained by step (2) is crossed into IDA-Ni affinity columns, and impurity elimination egg is first removed using Ni50 buffer solutions
In vain, afterwards using Ni500 buffer solutions elution destination protein；Elution gained destination protein then obtains Bt through desalting column PD-10 desalinations
Cry2Ab insecticidal proteins, Bt Cry2Ab insecticidal proteins are placed at -20 DEG C and preserved, it is standby.
Further, the formula of the Ni50 buffer solutions is NaCl 300mM, NaH2PO450mM, imidazoles 50mM；Ni500
The formula of buffer solution is NaCl 300mM, NaH2PO450mM, imidazoles 500mM.
Further, the 4 "-O-succinoyl ivermectin preparation process is：In molar ratio 1:3:6 weigh Yi Wei bacterium
Element, tert-butyl chloro-silicane and imidazoles, and be dissolved in tetrahydrofuran, stirring reaction 2 hours at room temperature, so as to Yi Wei
The 5-OH of rhzomorph is protected；Ivermectin after fetching protection is dissolved in dichloromethane, and adds DMAP, three
Ethamine and succinic anhydride, and protect after ivermectin, DMAP, triethylamine, succinic anhydride mol ratio be 1:
4:8:16, lucifuge water-bath afterwards flows back 3 hours, to carry out carboxylated transformation to the-OH of ivermectin 4 ", is then extracted and produced with ether
Thing, column chromatography for separation product；Then take the ivermectin after chromatography to be dissolved in methanol, and add p-methyl benzenesulfonic acid, and
The mol ratio of ivermectin and p-methyl benzenesulfonic acid after chromatography is 1:6, reaction is stirred at room temperature and is deprotected within 30 minutes, then
Adopt and product is extracted with ethyl acetate, last TLC separation produces 4 "-O-succinoyl ivermectins.
The present invention is it is also disclosed that application of the Bt Pesticidal toxins as agricultural fungicides.
The beneficial effects of the present invention are：A kind of Bt Pesticidal toxins of ivermectin coupling, Bt Pesticidal toxins tool are provided
There is stronger virulence, it has more preferable insecticidal effect to diamondback moth, can be used as agricultural fungicides, i.e., to preventing and treating agricultural pests
With more preferable application prospect；And the preparation method of the Bt Pesticidal toxins is disclosed simultaneously, the preparation method process is simple and convenient,
It is workable.
【Embodiment】
For a better understanding of the present invention, further illustrated with reference to embodiment and application examples in the explanation present invention
Hold, but these embodiments and application examples are only scopes that is exemplary, being not intended to limit the invention.And it should be noted that
Involved culture medium is existing routinely culture medium in the case of without specified otherwise in the present invention.
Embodiment 1
The preparation of 4 "-O-succinoyl ivermectins
In molar ratio 1:3:6 weigh ivermectin, tert-butyl chloro-silicane and imidazoles, and are dissolved in tetrahydrofuran,
Stirring reaction 2 hours at room temperature, so as to be protected to the 5-OH of ivermectin；Ivermectin after fetching protection is dissolved in two
In chloromethanes, and DMAP, triethylamine and succinic anhydride are added, and ivermectin, 4- dimethylaminos after protection
Pyridine, triethylamine, the mol ratio of succinic anhydride are 1:4:8:16, lucifuge water-bath afterwards flow back 3 hours, with to ivermectin 4 "-
OH carries out carboxylated transformation, then extracts product, column chromatography for separation product with ether；Then the ivermectin after chromatography is taken
It is dissolved in methanol, and adds p-methyl benzenesulfonic acid, and ivermectin after chromatography and the mol ratio of p-methyl benzenesulfonic acid is 1:
6, reaction is stirred at room temperature and is deprotected and (takes off hydroxyl protection) within 30 minutes, then adopt and product is extracted with ethyl acetate, it is last thin
Layer chromatography, which separates, produces 4 "-O-succinoyl ivermectins.
The structure of 4 "-O-succinoyl ivermectins is identified by MS and NMR technology：
1H NMR(400MHz,CDCl3)：δ 5.88 (d, J=8.7Hz, 1H), 5.80-5.69 (m, 2H), 5.39 (dd, J=
18.3,9.9Hz, 3H), 5.31 (s, 1H), 5.15 (d, J=13.2Hz, 1H), 5.00 (d, J=9.9Hz, 1H), 4.79 (s,
1H), 4.71 (d, J=10.2Hz, 3H), 4.30 (s, 1H), 4.15 (s, 1H), 3.98 (d, J=6.7Hz, 3H), 3.92-3.82
(m, 3H), 3.68 (d, J=12.7Hz, 5H), 3.66-3.61 (m, 2H), 3.49 (s, 1H), 3.41 (d, J=26.5Hz, 7H),
3.26 (dd, J=19.9,11.1Hz, 4H), 2.74-2.62 (m, 5H), 2.54 (s, 2H), 2.39-2.20 (m, 7H), 2.09-
1.96 (m, 6H), 1.89 (s, 5H), 1.78 (d, J=11.6Hz, 3H), 1.67 (d, J=12.7Hz, 5H), 1.63-1.47 (m,
15H), 1.27 (d, J=4.7Hz, 9H), 1.17 (dd, J=10.7,6.7Hz, 8H), 1.04-0.71 (m, 14H)；
ESI-MS(m/z):997.39[M+Na]+。
So that it is determined that the chemical constitution of the 4 "-O-succinoyl ivermectins, its chemical constitution are as follows：
Embodiment 2
The insecticidal proteins of thuringiensis are the preparation of Bt Cry2Ab insecticidal proteins
(1) cry2Ab genes are converted to colibacillus engineering to obtain containing the big of cry2Ab genes by thermal shock method
Enterobacteria engineering bacteria, the colibacillus engineering for containing cry2Ab genes is seeded on LB solid mediums afterwards, by LB
Solid medium is placed in incubated 24h at 30 DEG C and carries out actication of culture；
(2) the colibacillus engineering strain after activation is inoculated in LB fluid nutrient mediums, is placed in shaking flask 37 DEG C
Fermented and cultured, speed setting 180r/min, treat bacterium solution OD600After reaching 0.5,25 DEG C are adjusted to, 180r/min continues
Cultivate 24h；Then zymotic fluid obtained by fermented and cultured is placed in 4 DEG C, centrifuges 10min under the conditions of 10000r/min, after centrifugation terminates
Precipitation is taken to be resuspended in Na2CO3Lysate, the ultrasonic degradation 30min at 4 DEG C, 4 DEG C of solution, 10000r/min centrifugations after cracking
30min, take supernatant；
(3) supernatant obtained by step (2) is crossed into IDA-Ni affinity columns, first using Ni50 buffer solutions (NaCl 300mM,
NaH2PO450mM, imidazoles 50mM) remove foreigh protein removing；Ni500 buffer solutions (NaCl 300mM, NaH are used afterwards2PO450mM, miaow
Azoles 500mM) elution destination protein；Elution gained destination protein then obtains Bt Cry2Ab desinsection eggs through desalting column PD-10 desalinations
In vain, Bt Cry2Ab insecticidal proteins are placed at -20 DEG C and preserved, it is standby.
Embodiment 3
The preparation of Bt Pesticidal toxins
Example 1 prepares 4 "-O-succinoyl ivermectins (0.5mmol) of gained, is dissolved with 1mL DMSO, and
EDC (0.75mmol) and NHS (0.75mmol) is added, 2h is stirred at room temperature, the carboxyl of 4 "-O-succinoyl ivermectins is entered
Row activation, 4 "-O-succinoyl ivermectins after must activating are stand-by；Example 2 prepares the Bt Cry2Ab desinsections of gained
Albumen configures the Na of the Cry2Ab insecticidal proteins of Bt containing 5mmol/L2CO3/NaHCO3Buffer solution, i.e. Bt Cry2Ab insecticidal proteins are molten
Liquid, it is stand-by；Afterwards under the conditions of 4 DEG C, 4 "-O-succinoyl ivermectins after 100 μ L are activated add the Bt
In Cry2Ab insecticidal proteins solution 1mL, stir 1 hour and carry out coupling reaction, obtain Bt Pesticidal toxins.
Application examples 1
The insecticidal activity analysis of Bt Pesticidal toxins
(1) preparation of diamondback moth
The pickles chrysalis of indoor population is gathered, is sprouted wings, collects Adult worms producting eggs, an Eggs of Diamondback Moth is collected within every 24 hours, receives
The Eggs of Diamondback Moth of same batch of collection is raised in case LC in growth cabinet with similarity condition50Measure, the condition of the growth cabinet
For：25 DEG C of temperature, relative humidity 70%, photoperiod 16：8(L：D).
(2) verification method and result
The Bt that gained is prepared using 24 hole plate feeding methods measure Bt Cry2Ab insecticidal proteins, Acetamiprid and embodiment 3 is killed
Worm poison element sets experimental group and control group to the death rate of diamondback moth second instar larvae.Specific experiment method is experimental group：
Bt Pesticidal toxins are diluted, obtain the Bt Pesticidal toxins solution of various concentrations；Then in an aseptic environment, draw 1mL's
Feed is sub-packed in 24 orifice plates and naturally dry, the Bt Pesticidal toxins for drawing the various concentrations that 100 μ L have been configured respectively afterwards are molten
Liquid is simultaneously uniformly coated on feed surface, with 100 μ L Na2CO3/NaHCO3Buffer solution as blank control, each group carry out mark,
Dried under nature；Two age diamondback moths are inoculated into 24 orifice plates () per hole 5-7 only, and each hole of concentration gradient 4 repeats, and survey within 48 hours
The death rate (diamondback moth is motionless to be defined to be touched with writing brush for the death of worm) of diamondback moth, draws LC50 after fixed.In control group, with reality
The unique difference for testing group is to be used as effector using Bt Cry2Ab insecticidal proteins.Result of the test see the table below 1.
The insecticidal effect of the Bt Pesticidal toxins of table 1
Analyzed, drawn via table 1 and using the regression model of SPSS softwares：Control group is Bt Cry2Ab desinsection eggs
The LC50 to two age diamondback moths is 0.922 μ g/cm in vain2, experimental group is LC50 of the Bt Pesticidal toxins of the present invention to two age diamondback moths
For 0.652 μ g/cm2, insecticidal toxicity probably improves 1.41 times of this results and shows that Bt Pesticidal toxins of the present invention can actually carry
The high insecticidal toxicity to diamondback moth.
In summary, the present invention has stronger virulence, Bt desinsections of the present invention by coupling and the Bt Pesticidal toxins formed
Toxin has more preferable insecticidal effect to diamondback moth, and in other words, it can be used as agricultural fungicides to be used to murder diamondback moth, right
Preventing and treating agricultural pests have more preferable application prospect.
Claims (5)
- A kind of 1. Bt Pesticidal toxins of ivermectin coupling, it is characterised in that：The concrete operation step of its preparation method is as follows：Press 1:1.5:1.5 mol ratio weighs 4 "-O-succinoyl ivermectins, EDC and NHS respectively, and is dissolved in DMSO solvents, with The carboxyl of 4 "-O-succinoyl ivermectins is activated, 4 "-O-succinoyl ivermectins after must activating are stand-by； Take the Na of the Bt Cry2Ab insecticidal proteins configuration Cry2Ab insecticidal proteins of Bt containing 5mmol/L2CO3/NaHCO3Buffer solution, i.e. Bt Cry2Ab insecticidal proteins solution, it is stand-by；Measure respectively 4 "-O-succinoyl ivermectins after the activation prepared with it is described Bt Cry2Ab insecticidal proteins solution, stir 1 hour and carry out coupling reaction, obtain Bt Pesticidal toxins.
- A kind of 2. Bt Pesticidal toxins of ivermectin coupling according to claim 1, it is characterised in that：The Bt The preparation process of Cry2Ab insecticidal proteins is as follows：(1) cry2Ab genes are converted to colibacillus engineering to obtain the large intestine bar containing cry2Ab genes by thermal shock method Bacterium engineering bacteria, it is seeded to afterwards on LB solid mediums, LB solid mediums are placed in into incubated 24h at 30 DEG C is carried out Actication of culture；(2) the colibacillus engineering strain after activation is inoculated in LB fluid nutrient mediums, is placed in 37 DEG C of fermentations in shaking flask Culture, speed setting 180r/min, treats bacterium solution OD600After reaching 0.5,25 DEG C are adjusted to, 180r/min continues to cultivate 24h；Then zymotic fluid obtained by fermented and cultured is placed in 4 DEG C, centrifuges 10min under the conditions of 10000r/min, it is heavy that centrifugation takes after terminating Shallow lake is resuspended in Na2CO3Lysate, the ultrasonic degradation 30min at 4 DEG C, 4 DEG C of solution, 10000r/min centrifugation 30min after cracking, Take supernatant；(3) supernatant obtained by step (2) is crossed into IDA-Ni affinity columns, and foreigh protein removing is first removed using Ni50 buffer solutions, after Destination protein is eluted using Ni500 buffer solutions；Elution gained destination protein then obtains Bt Cry2Ab through desalting column PD-10 desalinations Insecticidal proteins, Bt Cry2Ab insecticidal proteins are placed at -20 DEG C and preserved, it is standby.
- A kind of 3. Bt Pesticidal toxins of ivermectin coupling according to claim 2, it is characterised in that：The Ni50 bufferings The formula of liquid is NaCl 300mM, NaH2PO450mM, imidazoles 50mM；The formula of Ni500 buffer solutions be NaCl 300mM, NaH2PO450mM, imidazoles 500mM.
- A kind of 4. Bt Pesticidal toxins of ivermectin coupling according to claim 1, it is characterised in that：The 4 "-O- Succinoyl ivermectin preparation process is：In molar ratio 1:3:6 weigh ivermectin, tert-butyl chloro-silicane and miaow Azoles, and be dissolved in tetrahydrofuran, stirring reaction 2 hours at room temperature, so as to be protected to the 5-OH of ivermectin；Fetch protection Ivermectin afterwards is dissolved in dichloromethane, and adds DMAP, triethylamine and succinic anhydride, and after protection Ivermectin, DMAP, triethylamine, the mol ratio of succinic anhydride are 1:4:8:16, the backflow 3 of lucifuge water-bath afterwards is small When, to carry out carboxylated transformation to the-OH of ivermectin 4 ", then extract product, column chromatography for separation product with ether；Then layer is taken Ivermectin after analysis separation is dissolved in methanol, and adds p-methyl benzenesulfonic acid, and the ivermectin after chromatography with to first The mol ratio of benzene sulfonic acid is 1:6, reaction is stirred at room temperature and is deprotected within 30 minutes, then adopt and product is extracted with ethyl acetate, finally TLC separation produces 4 "-O-succinoyl ivermectins.
- 5. the application for the Bt Pesticidal toxins that ivermectin described in a kind of claim 1 is coupled, it is characterised in that：The Bt desinsections poison Application of the element as agricultural fungicides.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113817008A (en) * | 2021-07-15 | 2021-12-21 | 湖州师范学院 | Preparation method and application of novel succinyl sixteen-membered macrolide |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006007595A2 (en) * | 2004-07-01 | 2006-01-19 | E.I. Dupont De Nemours And Company | Synergistic mixtures of anthranilamide invertebrate pest control agents |
| CN101029067A (en) * | 2006-03-01 | 2007-09-05 | 中国农业科学院兰州畜牧与兽药研究所 | Synthesis of ivermectin artificial antigen |
| CN102675411A (en) * | 2012-05-08 | 2012-09-19 | 福建省农业科学院农业生物资源研究所 | Method for preparing biotoxin |
| CN103228670A (en) * | 2010-12-13 | 2013-07-31 | 先正达参股股份有限公司 | Cry1I proteins and genes for insect control |
-
2017
- 2017-09-21 CN CN201710859935.9A patent/CN107417774B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006007595A2 (en) * | 2004-07-01 | 2006-01-19 | E.I. Dupont De Nemours And Company | Synergistic mixtures of anthranilamide invertebrate pest control agents |
| CN101029067A (en) * | 2006-03-01 | 2007-09-05 | 中国农业科学院兰州畜牧与兽药研究所 | Synthesis of ivermectin artificial antigen |
| CN103228670A (en) * | 2010-12-13 | 2013-07-31 | 先正达参股股份有限公司 | Cry1I proteins and genes for insect control |
| CN102675411A (en) * | 2012-05-08 | 2012-09-19 | 福建省农业科学院农业生物资源研究所 | Method for preparing biotoxin |
Non-Patent Citations (1)
| Title |
|---|
| PAN Z等: "Characterization of a new cry2Ab gene of Bacillus thuringiensis with high insecticidal activity against Plutella xylostella L.", 《 WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113817008A (en) * | 2021-07-15 | 2021-12-21 | 湖州师范学院 | Preparation method and application of novel succinyl sixteen-membered macrolide |
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